Single-Molecule Platform For Drug Discovery: Methods And Apparatuses For Drug Discovery, Including Discovery Of Anticancer And Antiviral Agents

ABSTRACT

The present application discloses methods and apparatuses for single molecule drug screening, discovery and validation. These methods and apparatuses allow a user to detect rapidly, using observation of single molecules, whether and how a drug candidate interferes with a target enzyme involved in a particular disease pathway. The methods and apparatuses described herein utilize single molecule manipulation and detection technologies (e.g., optical or magnetic tweezers) to directly detect whether the characteristic dynamics, or “mechanical signature,” of the target enzyme-substrate interaction are substantially altered or modulated by a drug candidate. Furthermore, the methods and apparatuses are useful for analyzing the modulation of the mechanical signature in order to identify potential interference mechanisms of a drug candidate. In one aspect of the invention, the methods and apparatuses disclosed herein relate to monitoring the real-time dynamic mechanical signatures of individual polymerase molecules (e.g. DNA polymerases, RNA polymerases, and reverse transcriptases) along a polynucleotide substrate in the presence of drug candidates that either inhibit or otherwise modulate the polymerization process. Identification and analysis of such drug candidates is critical for anti-viral, anti-cancer, and antibiotic drug development.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/790,071, filed on Apr. 23, 2017 and claims the benefit of U.S.Provisional Application No. 60/793,720, filed on Apr. 21, 2006, theentire teachings of which are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to the screening and validation of drug candidatesthat target enzymes, including DNA polymerases, RNA polymerases, andreverse-transcriptases.

BACKGROUND OF THE INVENTION

Approximately thirty percent of drugs in clinical use inhibit adisease-related enzymatic process (Copeland, R. A. Evaluation of enzymeinhibitors in drug discovery: a guide for medicinal chemists andpharmacologists. (Wiley-Interscience, 2005)). Thus, the discovery of newenzyme inhibitors is an important area of research in biochemistry andpharmacology.

Polymerase inhibitors are valuable in both clinical and researchsettings. These inhibitors help in elucidating the mechanistic aspectsof transcription and DNA replication, in mapping structure-functionrelationships, and in characterizing protein activity. Polymeraseinhibitors are also among the most attractive drug targets. Knowledgeabout these inhibitors, their structures, and their mechanisms enablethe design of new drugs such as anti-cancer agents, antiviral agents,and antibiotics that will be effective against new pathogens andantibiotic resistant mutants of known pathogens. Because some of theseinhibitors have been reported to induce and/or inhibit apoptosis, theyalso provide valuable tools for investigating apoptosis. Likewise,because some of these agents block specific steps of DNA transcription,polymerase inhibitors can help to elucidate the role of transcriptionalcontrol in regulating the expression of target genes in various healthyand disease states.

Drugs that target polymerase proteins involved in particular diseasepathways are well known in the art. Reverse transcriptase inhibitors(RTIs), for example, are a class of antiretroviral drugs that targetconstruction of viral DNA by inhibiting the activity of reversetranscriptase.

There are two subtypes of RTIs with different mechanisms of action:nucleoside and nucleotide analogue RTIs are incorporated into the viralDNA leading to chain termination, while non-nucleoside-analog RTIs actas competitive inhibitors of the reverse transcriptase enzyme. CurrentAIDS therapeutics that function by inhibiting HIV reverse transcriptaseare described in the art (see, e.g., Bean et al., Appl Environ Microbiol72:5670-5672 (2005)), and include Efavirenz (brand names SUSTIVA® andSTOCRIN®) and Nevirapine (also marketed under the trade name VIRAMUNE®).Antibiotics that target polymerase proteins (e.g. rifampin) and cancerdrugs that target polymerase proteins (e.g. cisplatin) are also known inthe art.

Many drugs have been found to be efficacious in the treatment of cancer.These include diverse chemical compounds such as antimetabolites (e.g.,methotrexate and fluorouracil), DNA-damaging agents (e.g.,cyclophosphamide, cisplatin, and doxorubicin), mitotic inhibitors (e.g.,vincristine), nucleotide analogues (e.g., 6-mercaptopurine), inhibitorsof topoisomerases involved in DNA repair (e.g., etoposide), inhibitorsof DNA polymerase (e.g., bleomycin), and intercalating agents likemitoxantrone.

Several drugs targeting enzymes of mammalian DNA replication arecurrently being investigated as promising candidates for cancerchemotherapy or as probes for understanding. the roles of specificenzymes in DNA replication and repair. These potential drug candidatesinclude corylifolin, bakuchiol, resveratrol, Neobavaisoflavone, anddaidzein (see Sun et al., J. Nat. Prod. 61, 362-366 (1998)).

Other examples of DNA and RNA polymerase inhibitors include ActinomycinD, Streptomyces sp.; a-Amanitin, Amanita sp.; Aphidicolin, HSYreplication inhibitor, BPS; Methyl a-Amanitin Oleate; Novobiocin, SodiumSait; Rifampicin; RNA Polymerase III Inhibitor; and Actinomycin D,7-Amino. Three polymerase inhibitors currently in Phase II trials foruse against Hepatitis C Virus are Idenix/Novartis' valopicitabine(NM283); ViroPharma's HCV-796; and Roche's R1 626. Roche/Idenix are alsoinvestigating valtorcitabine (val-LdC), a first strand viral DNAsynthesis inhibitor in Phase II HCV trials after initial success as anHBV treatment.

DNA damaging agents provide some of the most successful treatments forcancer. The enzyme Poly(ADP-ribose)polymerase (i.e. PARP) can helprepair DNA damage caused by the DNA damaging agents used to treatcancer. As PARP activity is often increased in cancer cells, it providesthese cells with a survival mechanism. ABT-888 (Abott Oncology), forexample, is an oral PARP-inhibitor developed by Abbott to prevent DNArepair in cancer cells and increase the effectiveness of common cancertherapies such as radiation and alkylating agents. Moreover, preclinicaldata indicates ABT-888 has improved the effectiveness of radiation andmany types of chemotherapy in animal models of cancer.

These selected publications from the last 5 years illustrate the currentstate of the artwith regard to the activity, mechanisms, andbiochemistry of polymerase inhibitors:

-   Brown J A, Duym W W, Fowler J D, Suo, Z. (2007) “Single-turnover    Kinetic Analysis of the Mutagenic Potential of    8-Oxo-7,8-dihydro-2′-deoxyguanosine during Gap-filling Synthesis    Catalyzed by Human DNA Polymerases lambda and beta.” J Mol Biol.    [Epub ahead of print] Suo, Z., Abdullah M A. (2007) “Unique    Composite Active Site of the Hepatitis C Virus NS2-3 Protease: aNew    Opportunity for Antiviral Drug Design.” ChemMedChem. 2(3), 283-284.    Roettger M P, Fiala K A, Sompalli S, Dong Y, Suo Z. (2004)    “Pre-steady-state kinetic studies of the fidelity of human DNA    polymerase mu”, Biochemistry 43(43), 13827-38.-   Fiala K A, Abdel-Gawad W, Suo Z. (2004) “Pre-steady-state kinetic    studies of the fidelity and mechanism of polymerization catalyzed by    truncated human DNA polymerase lambda.”, Biochemistry 43(21),    6751-62.-   Fiala, K. A & Suo Z.* (2004) Pre-Steady State Kinetic Studies of the    Fidelity of Sulfolobus solfataricus P2 DNA Polymerase IV.    Biochemistry 43, 2106-2115-   Fiala, K. A & Suo Z.* (2004) Mechanism of DNA Polymerization    Catalyzed by Sulfolobus solfataricus P2 DNA Polymerase IV.    Biochemistry 43, 2116-2125-   Fiala, K. A, Abdel-Gawad, W. & Suo Z.* (2004) Pre-Steady-State    Kinetic Studies of the Fidelity and Mechanism of Polymerization    Catalyzed by Truncated Human DNA Polymerase Lambda. Biochemistry,    accepted and in press.-   Allison, A. J., Ray, A., Suo Z., Colacino, J. M., Andeson, K. S.,    Johnson, K. A. (2001) “Toxicity of Antiviral Nucleoside Analogs and    the Human Mitochondrial DNA Polymerase”, J. Biol. Chem. 276,    40847-40857.

New drugs are the products of a long and involved drug developmentprocess, the first step of which is the discovery of compounds withpromising activity. New enzyme inhibitors can be discovered by screeninglibraries of drug candidate compounds against a target enzyme.Conventional drug screening and validation approaches utilize micro- tomilli-scale biochemical or cellular assays to detect downstreambiochemical or cellular signatures of enzymatic interference. In view ofthe limitations of conventional drug screening methods, there remains aneed in the art for improved methods and apparatuses for the detectionof promising drug candidates.

SUMMARY OF THE INVENTION

The present application discloses methods and apparatuses for singlemolecule drug screening, discovery and validation. These methods andapparatuses allow a user to detect rapidly, using observation of singlemolecules, whether and how a drug candidate interferes with a targetenzyme involved in a particular disease pathway. The methods andapparatuses described herein utilize single molecule manipulation anddetection technologies (e.g., optical or magnetic tweezers) to directlydetect whether the characteristic dynamics, or “mechanical signature,”of the target enzyme-substrate interaction are substantially altered ormodulated by a drug candidate. Furthermore, the methods and apparatusesare useful for analyzing the modulation of the mechanical signature inorder to identify potential interference mechanisms of a drug candidate.

In one aspect of the invention, the methods and apparatuses disclosedherein relate to monitoring the real-time dynamic mechanical signaturesof individual polymerase molecules (e.g. DNA polymerases, RNApolymerases, and reverse transcriptases) along a polynucleotidesubstrate in the presence of drug candidates that either inhibit orotherwise modulate the polymerization process. Identification andanalysis of such drug candidates is critical for anti-viral,anti-cancer, and antibiotic drug development.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates exemplary flow charts of a method aspect of thisinvention for screening multiple drug candidates that target aparticular enzyme.

FIGS. 2A-2E illustrates exemplary mechanical signatures from DNApolymerase in the presence of various drug candidates (such aspolymerase inhibitors).

FIG. 2A shows a representative signal expected from a control samplewhere no polymerase inhibitor is present. In this scenario, thepolymerase binds to single stranded (ss) DNA and converts this templateinto double stranded (ds) DNA, steadily shortening the overall templatelength with time. FIG. 2B shows an exemplary mechanical signatureexpected from an optical tweezers apparatus that would indicate that adrug candidate has slowed the replication process. Note that the overallslope of the template length plot, which corresponds to thepolymerization velocity, has lessened. FIG. 2C shows an exemplarymechanical signature from an optical tweezers apparatus that indicatesthat a drug candidate has induced abortive transcription/prematuretermination, wherein the drug candidate has derailed or stalled thereplication process before its natural completion point. FIG. 2D showsan exemplary mechanical signature from an optical tweezers apparatusthat indicates that initiation of DNA replication is inhibited by thedrug candidate. FIG. 2E shows an exemplary mechanical signature from anoptical tweezers apparatus that indicates the drug candidate induces thepolymerase enzyme to operate in an exonucleolysis mode, wherein itexcises bases rather than polymerizes base-pairs. This signature wouldlikely only occur in polymerases with an active exonucleolysis, or“proof-reading” site.

FIG. 3 illustrates exemplary flow charts of a method aspect of thisinvention for screening multiple drug candidates that target aparticular polymerase enzyme, including DNA and RNA polymeraseinhibitors and reverse transcriptase inhibitors.

FIG. 4A illustrates exemplary experimental geometries for obtaining, viaan optical tweezers based single-molecule measurement system, themeasurement of a polymerase's position along a nucleic acid templatetemplate. Here a DNA molecule is held immobilized and stretched betweentwo plastic latex beads, as the polymerase moves along the DNA template.In the case of DNA replication, the distance between the two beadsdecreases at a given force as the template DNA is converted from singleto double stranded DNA. The methods of attachment of the nucleic acid tothe beads can be via streptavidin-biotin, or dig-anti-dig, or othercovalent linkages.

FIG. 4B illustrates additional exemplary experimental geometries forobtaining, via an optical tweezers-based single-molecule measurementsystem, the length of a nucleic acid as it is processed by a polymeraseenzyme. Here a DNA molecule is held immobilized and stretched between aplastic latex bead and a streptavidin-coated (triangles) glass surface.As the polymerase moves along the DNA template in DNA replication, thelength of the the DNA tether decreases at a given force as theimmobilized template DNA is converted from single to double strandedDNA.

FIG. 4C illustrates an embodiment of the present invention where thenucleic acid template is attached to a dielectric bead that is held by asecond, immobilized optical trap, shown at left, that exerts a strongtrapping force that is much greater than the force exerted by thepolymerase on the nucleic acid template. Here the polymerase is attachedby the relevant affinity chemistry to the other bead.

FIG. 4D demonstrates how the nucleic acid can be immobilized to a rigidsurface, such as a cover slip or microwell plate, via complementaryfunctionalization of the nucleic acid template and the rigid surface,while again the polymerase is attached to the second bead.

FIG. 5A illustrates schematically the single molecule detection systemwhich includes a representative optical tweezers apparatus thatcomprises trapping, force detection, beam steering and isotensioncapabilities and force-feedback optical trapping subsystem thatmaintains a constant force on a trapped bead, even as the beadexperiences other forces due to the enzymatic activity of a polymerase.

FIG. 5B illustrates the detection system in the single molecule trackingsystem by which a quadrant photodiode is utilized to detect thedisplacement of a dielectric sphere from an optical trap's center. Theposition of the bead with respect to the optical trap is recorded ontothis quadrant photodetector. Deviations from this center of the opticalwell are used to quantify the picoNewton sized forces acting on thebead.

FIGS. 6A and 6B illustrate data demonstrating the capability to observethe dynamics of the DNA polymerase motor with an optical tweezersapparatus as it moves forwards (polymerization, FIG. 6A and backwards(exonuclease activity, FIG. 6B) along a DNA template. A velocity ofabout 0.5 microns/minute corresponds to about 25 base-pairspolymerized/second. In FIGS. 6A and 6B, position of the polymerase alongDNA is shown as a function of time. The slope gives the rate of changein length of the DNA template at a given force while it is beingreplicated. This is just the single molecule velocity of the polymerasemotor.

FIG. 7 illustrates the ability to probe even fine-structure dynamics ofDNA polymerization. To achieve this level of resolution, anacousto-optic deflector system is preferably utilized to achieveisotension conditions along the DNA strand during polymerization. InFIG. 7, the extension (μm) of the DNA is shown versus time (min). In theleft plot, the black line shows full data set, including relaxation dueto flow (left boxed region) before the contraction (right hand boxedregion) begins. At right, extension versus time is shown for thecontraction region. The raw data line shows original length-vs-timedata, while the smoothed line shows 100-pt adjacent average of thisdata. The straight line indicates the average slope through thiscontraction region, about 300 hp/second.

FIG. 8 illustrates key features of the conventional approach to drugscreening and discovery.

FIG. 9 illustrates key features of the novel drug screening anddiscovery techniques described herein.

FIG. 10 illustrates some of the advantages of the novel drug screeningand discovery techniques described herein as compared to conventionalapproaches to drug screening and discovery.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

DNA and RNA polymerase and reverse transcriptase (RT) inhibitors arevaluable in both clinical and research settings. These inhibitors helpin elucidating the mechanistic aspects of transcription and DNAreplication, in mapping structure-function relationships, and incharacterizing protein activity. These polymerase and RT inhibitors arealso among the most attractive drug targets. Knowledge about theseinhibitors, their structures, and their mechanisms enable the design ofnew drugs such as anti-cancer agents and antibiotics that will beeffective against new pathogens and antibiotic-resistant mutants ofknown pathogens. Because some of these inhibitors have been reported toinduce and/or inhibit apoptosis, they also provide valuable tools forinvestigating apoptosis. Likewise, because some of these agents blockspecific steps of DNA transcription, polymerase inhibitors can help toelucidate the role of transcriptional control in regulating theexpression of target genes in various healthy and disease states.

Drugs that target polymerase proteins involved in a particular diseasepathway are well known in the art. Reverse transcriptase inhibitors(RTIs), for example, are a class of antiretroviral drugs that targetconstruction of viral DNA by inhibiting the activity of reversetranscriptase. There are two subtypes of RTIs with different mechanismsof action: nucleoside and nucleotide analogue RTIs are incorporated intothe viral DNA leading to chain termination, whilenon-nucleoside-analogue RT is act as competitive inhibitors of thereverse transcriptase enzyme. Current AIDS therapeutics that function byinhibiting HIV reverse transcriptase are described in the art (see,e.g., Bean et al., Appl Environ Microbiol 72:5670-5672 (2005)), andinclude Efavirenz (brand names SUSTIVA® and STOCRJN®) and Nevirapine(also marketed under the trade name VIRAMUNE®).

Many drugs have been found to be efficacious in the treatment of cancer.These include diverse chemical compounds such as antimetabolites (e.g.,methotrexate and fluorouracil), DNA-damaging agents (e.g.,cyclophosphamide, cisplatin, and doxorubicin), mitotic inhibitors (e.g.,vincristine), nucleotide analogues (e.g., 6-mercaptopurine), inhibitorsof topoisomerases involved in DNA repair (e.g., etoposide), inhibitorsof DNA polymerase (e.g., bleomycin), or intercalating agents likemitoxantrone.

Several drugs targeting enzymes of mammalian DNA replication arecurrently being investigated as promising candidates for cancerchemotherapy or as probes for understanding the roles of specificenzymes in DNA replication and repair. These potential drug candidatesinclude, but are not limited to corylifolin, bakuchiol, resveratrol,Neobavaisoflavone, and daidzein (see Sun et al., J. Nat. Prod. 61,362-366 (1998).

Other examples of DNA and RNA Polymerase Inhibitors include: ActinomycinD, Streptomyces sp.; a-Amanitin, Amanita sp.; Aphidicolin; HSVReplication Inhibitor, BPS; Methyl a-Amanitin Oleate; Novobiocin, SodiumSalt; Rifampicin; RNA Polymerase III Inhibitor; Actinomycin D, 7-Amino.Three polymerase inhibitors currently in Phase II trials for use againstHepatitis C Virus are Idenix/Novartis' valopicitabine (NM283);ViroPhanna's HCV-796 and Roche's R1626. Roche/Idenix are alsoinvestigating valtorcitabine (val-LdC)—a first strand viral DNAsynthesis inhibitor in Phase II HCV trials after initial success as anHBV treatment.

DNA damaging agents provide some of the most successful treatments forcancer. The enzyme Poly (ADP-ribose)polymerase (i.e. PARP) can helprepair DNA damage caused by the DNA damaging agents used to treat cancera. As PARP activity is often increased in cancer cells, it providesthese cells with a survival mechanism. ABT-888, for example, is an oralPARP-inhibitor developed by Abbott Oncology to prevent DNA repair incancer cells and increase the effectiveness of common cancer therapiessuch as radiation and alkylating agents. Moreover, preclinical dataindicates ABT-888 has improved the effectiveness of radiation and manytypes of chemotherapy in animal models of cancer.

New drugs are the products of a long and involved drug developmentprocess, the first step of which is the discovery of compounds withpromising activity. New enzyme inhibitors can be discovered by screeninglibraries of drug candidate compounds against a target enzyme. Drugcandidates include compounds found in nature, compounds synthesized bycombinatorial chemistry approaches, and compounds created via rationaldrug design. Conventional drug screening and validation approachesutilize micro- to milliscale biochemical or cellular assays to detectdownstream biochemical or cellular signatures of enzymatic interference.For example, an assay might, via radioactive labeling, measure anychanges in the quantity of a reaction product whose synthesis iscatalyzed by the target enzyme.

Single-molecule techniques offer several key benefits over conventionalin vitro assay methods for drug screening, as they use less reagents andoffer much more detail into the mechanism of drug action on the target.For example, single molecule techniques enable transient states to beobserved, thereby making it possible to selectively screen for chemicalcompounds that isolate these steps. Single molecule approaches thusenable the identificiation, testing, and validation of polymerase orenzyme inhibitors that target key phases in biochemical processes, e.g.,transcription or replication initiation. Many biochemical processesconsist of multiple transient steps, such as promoter binding,initiation, elongation, and termination in transcription. Because thetotal number of potential drug targets can be extremely high, singlemolecule approaches provide a critical advantage in speeding up theprocess of drug screening and discovery by focusing the efforts early onto only those steps of the process that are most affected by the drugcandidate.

By elucidating the kinetic mechanisms of enzymes involved in DNA/RNAreplication and repair, antiviral and anti-cancer drug candidates can beidentified based on rational drug design. Kinetic studies use a varietyof pre-steady state kinetic methods including rapid chemical quench-flowand stopped-flow techniques. These methods allow reactions to bequenched in milliseconds, and provide more kinetic information than thetraditional steady-state kinetic methods. Single molecule techniqueselucidate the elementary steps of reactions occurring at the activesites of enzymes and can significantly enhance rational drug design.

Fears about the possible release of smallpox by bioterrorists have ledto intensive efforts to find an effective molecule to inhibit viralinfection which does not yet exist. Since smallpox virus (variola virus)and the smallpox vaccine (vaccinia virus) are highly homologous, thelatter has been used as a very good surrogate model. Vaccinia virus DNApolymerase, for instance, is about 99% identical to in the polymerase inthe smallpoxvirus.

The Hepatitis C virus has infected at least 2-3% of human population.Viral genome replication has been intensively studied. The RNA-dependentRNA polymerase, NS5B, is central to viral replication, and is a majorantiviral drug target. Although there are extensive biochemical andsteady-state kinetic studies on this polymerase, the elementary steps ofnucleotide incorporation catalyzed by NS5B are still undefined. Theseinvestigations enable the rational design of nucleoside inhibitors.

In the last decade, new tools (for example optical tweezers,atomic-force-microscopy, and small glass fibers) have been developed tomanipulate small objects and also to investigate the forces involved inthe systems studied (see, e.g., Smith et al., Science 271:795 (1996) andCluzel, et al., Science 271, 792-794 (1996)). In particular, optical ormagnetic “tweezers” or “traps” trap particles with forces generated byoptical intensity gradients, and can be used to manipulate and studymicroscopic molecules at the single-molecule level. Optically generatedforces strong enough to form a three-dimensional trap can be obtained bybringing a laser beam with an appropriately shaped wavefront to a tightfocus with a high numerical aperture lens. The principles of opticaltrapping are well known in the art and are summarized in, for example,Neuman and Block, Rev. Sci. Instr. 75:2787-2809 (2004).

In the biological sciences, optical tweezers have been used to measuredisplacements in the nm range of molecules ranging in size from 10 nm toover 100 mm. Common to most optical tweezers biophysical experiments isthe attachment of dielectric beads to biological molecules (e.g.substrates and/or enzymes), so that the biological molecules can bemanipulated by the optical trap and mechanical measurements can betaken. Various biochemical and molecular biology methods are known inthe art for attaching nucleic acids, other substrates, enzymes and otherbiomacromolecules to functionalized surfaces and beads. For example, DNAcan be labeled with biotin moieties that will bind to commerciallyavailable, streptavidin-coated, micron-coated dielectric spheres (e.g.,from Bangs' Laboratories).

Two of the main uses for optical traps in biology have been the study ofthe physical properties of DNA, and the study of molecular motors suchas DNA and RNA polymerases ((see, e.g., Davenport et al., Science287:2497-2500 (2000); Maier et al., PNAS 97:12002-12007 {2000); Wang etal., Science 283:902-907 (1998); Wuite et al., Nature 404:103-106(2000); and Yin et al., Science 270:1653-1656 (1995)). For example,researchers have been able to measure the sequence-dependence of theforces necessary to “unzip” double-stranded DNA (Voulgarakis, et al.,Nano Letters 6, 1483-1486 (2006)). In addition, optical tweezers wereused to elucidate the mechanism whereby kinesin walks along amicrotubule (Kuo & Sheetz, Science 260,232 (1993) and Block, et al.,Nature 348, 348-352 (1990)). Quite recently, researchers detected withsingle base-pair resolution the stepping action of the RNA polymerasealong a molecule of DNA (Abbondanzieri, et al., Nature 438, 460-465(2005)). At Nanobiosym, high-resolution optical tweezers have beenutilized to experimentally demonstrate the role of various environmentalfactors on the dynamics of polymerases (Goel et al, NatureNanotechnology review article in press).

In all such studies, optical tweezers were utilized to directly measurethe mechanical dynamics of a substrate-enzyme interaction. In thesestudies, the details of experimental setup and measurements taken aredependent on the biological function of the enzyme and/or the substrateinvolved. For example, the mechanical measurements of interest mayinclude: the elasticity of substrate polymers, including stretching andrelaxation dynamics; the time dependent velocity of an enzyme that is“processing” a linear substrate, such as polymerase bound to a nucleicacid; the deformation of a substrate caused by enzymatic binding; and/orthe efficiency or accuracy of substrate binding and processing. Byintegrating position and/or force-sensing subsystems into an opticaltweezers apparatus, all such measurements are possible.

Novel methods and apparatuses for single molecule drug screeningdiscovery and validation are disclosed herein. These methods andapparatuses allow a user to detect rapidly, at the single-moleculelevel, whether and how a drug candidate interferes with anenzyme-substrate interaction involved in a particular disease pathway.In particular, interactions between candidate drugs and a single targetenzyme molecule can be observed. The methods and apparatuses describedherein utilize single molecule manipulation technologies (e.g. opticalor magnetic tweezers or traps) to directly detect, at thesingle-molecule level, whether a drug candidate can mechanically orchemically alter the enzyme-substrate interaction.

In a preferred embodiment, the present methods and apparatuses can beutilized to quickly screen, test, and validate new drug candidates thatmodify, inhibit or otherwise interfere with polymerase enzymes such asDNA polymerase, RNA polymerase, and RNA reverse transcriptase, etc., andto better elucidate the mechanism whereby the polymerase/substrateinteraction is inhibited.

Normal enzymatic activity on a substrate produces a dynamic “mechanicalsignature.” The term “mechanical signature” as used herein refers to thebiomechanical trace of a single molecule of an enzyme as it interactswith it's substrate. The biomechanical trace can be measured usinginstruments, such as optical tweezers, that can detect displacements inthe nm range. As described above, this dynamic mechanical signature canbe determined by making a “mechanical measurement,” for example bymeasuring changes in the elasticity of substrate polymers, includingstretching and relaxation dynamics; the time-dependent velocity of anenzyme that is “processing” a linear substrate, such as polymerase boundto a nucleic acid; the deformation of a substrate caused by enzymaticbinding; and/or the efficiency or accuracy of substrate binding andprocessing.

The term “mechanical measurement” as used herein means a measurement ofthe mechanical dynamics of a substrate-enzyme interaction, wherein themechanical measurement detects the mechanical signature of a singlemolecule of a target enzyme and/or a single molecule of a substrate ofthe target enzyme. “Making a mechanical measurement” includes, forexample, measuring changes in the elasticity of substrate polymers,including stretching and relaxation dynamics; the time-dependentvelocity of an enzyme that is “processing” a linear substrate, such aspolymerase bound to a nucleic acid; the deformation of a substratecaused by enzymatic binding; and/or the efficiency or accuracy ofsubstrate binding and processing.

A preferred “mechanical measurement” is a measurement of the movement ofa reverse transcriptase, DNA polymerase, or RNA polymerase enzyme alonga polynucleotide (e.g. DNA or RNA) substrate. Thus, in particularembodiments of the method, discussed in more detail below, the real-timesingle molecule dynamics of a polymerase along a nucleic acid sequenceis monitored in the presence and absence of drug candidates via opticaltrapping techniques. Other mechanical measurements of apolymerase/substrate interaction which are not explicitly describedherein but are also measurable via single molecule detection techniques(e.g. optical tweezers) are also possible.

A mechanical measurement can be made in the presence or absence of adrug candidate. A “baseline mechanical signature” is a mechanicalsignature that was determined via a mechanical measurement that was madein the absence of a drug candidate.

The term “target” or “drug target” as used herein refers to abiomolecule that is involved in a disease pathway. Inhibiting orotherwise interfering with the activity of the target could bebeneficial in treating and/or preventing the disease. The term “targetenzyme” as used herein refers to an enzyme that is involved in a diseasepathway. Typically a target enzyme is a key enzyme involved in aparticular metabolic or signaling pathway that is specific to a diseasecondition or pathology, or to the infectivity or survival of a microbialpathogen. The “activity of a target enzyme” means the interaction of thetarget enzyme with a substrate of the target enzyme.

Target enzymes suitable for the present invention include enzymes thatbind to and interact with DNA and/or RNA. Examples include polymerases,such as DNA polymerases, RNA polymerases, and reverse transcriptases;topoisomerases; gyrases; exonucleases; and helicases. The target enzymescan be human enzymes, for example human enzymes involved in a diseasepathway such as cancer. In another embodiment, the target enzymes can beviral or bacterial enzymes, such as viral or bacterial enzymes involvedin viral- and/or bacterial mediated diseases. Other microbial enzymesare also contemplated as target enzymes suitable for the presentinvention.

A preferred target enzyme is a polymerase enzyme such as a DNApolymerase, an RNA polymerase, or a reverse transcriptase. Polymerasesinvolved in cancer pathways, especially human DNA polymerases involvedin human cancer pathways, are particularly preferred. Polymerasesinvolved in viral-mediated disease pathways, especially viral reversetranscriptases involved in viral-mediated disease pathways in humans(e.g. hepadnaviral reverse transcriptases such as Hepatitis B reversetranscriptase, and retroviral reverse transcriptases such as HIV-1reverse transcriptase) are also particularly preferred.

The present invention is also suitable for screening drug candidatesthat may interact with non-enzyme targets involved in disease pathways.Representative examples of non-enzyme targets are microtubules andribozymes (such as ribosomes). Ribosomes, in particular, use RNA as atemplate to build polypeptide chains, and thus can be thought of as agiant enzymes.

The term “substrate of the target enzyme” (or “enzyme substrate” or“substrate”) as used herein refers to a molecule upon which a targetenzyme acts. Enzymes catalyze chemical reactions involving one or moresubstrates. Enzyme substrates are well known in the art.

Preferred substrates include polymerase substrates, such aspolynudeotides (e.g. DNA and RNA). Polynucleotide substrates are alsoreferred to herein as polynucleotide or nucleic acid “templates.”Polynucleotide substrates can be double-stranded or single-stranded DNAor RNA sequences.

The term “drug candidate” or “candidate” as used herein refers to acompound that may inhibit or otherwise interfere with the activity of atarget, particularly a target enzyme. Drug candidates include compoundsfound in nature, compounds synthesized by combinatorial chemistryapproaches, and compounds created via rational drug design. Examples ofdrug candidates include compounds that interact with or may interactwith polynucleotides (e.g. DNA and/or RNA), and/or compounds thatinterfere with or may interfere with the activity of enzymes thatinteract with polynucleotides. Such compounds can be known or potentialDNA modifying agents, including DNA damaging agents (e.g. intercalatingagents that interfere with the structure of nucleic acids); DNA bendingagents; mismatch binding proteins; and/or alkylating agents.

In another embodiment, a drug candidate can be a compound that interactswith or may interact with a non-enzyme target involved in diseasepathway. Examples include compounds that interact with or may interactwith microtubules and/or ribosomes.

Some exemplary classes of drug candidates that can be probed via thepresent methods are described next. First, several classes of antibioticdrugs are suitable for interrogation by the present methods, includingdrugs that inhibit or otherwise interfere with the activity of bacterialpolymerases such as bacterial DNA polymerase, bacterial RNA polymerase(e.g., rifampin), and/or bacterial reverse-transcriptase. Second, thepresent methods could also be utilized to quickly screen, test, andvalidate new antiviral drug candidates that inhibit or somehow interferewith viral polymerases, especially viral reverse transcriptases (e.g.efavirenz and nevirapine), and to better elucidate the mechanism wherebythe RNA-reverse transcriptase interaction is inhibited. Third, severalclasses of anti-cancer drugs are also suitable for interrogation by thepresent methods. These include drugs that inhibit or otherwise interferewith the activity of DNA polymerases, RNA polymerases, topoisomerases,ribosomes, and/or microtubules (e.g. microtubule antagonists such asvincristine and taxol). Additionally, entirely new drug mechanisms,heretofore unknown, could be discovered and elucidated by the approachdescribed herein.

The term “single molecule detection apparatus” (or “single moleculedetection device”) as used herein refers to an apparatus that can beused to make a mechanical measurement of an enzyme-substrate interactionat the single-molecule level. Single molecule detection apparatusessuitable for the present invention include apparatuses used for magneticor optical trapping (e.g. optical tweezers), high-resolution fluorescentimaging coupled with quantum-dot labeling, and atomic force microscopy.Other apparatuses and variants of the apparatuses disclosed herein couldbe readily envisioned.

A preferred single molecule detection apparatus is an apparatuscomprising an optical trap or tweezers.

Instead of screening for a downstream effect of enzymatic interference,the methods described herein, dubbed NANO-VALID™, determine via direct,single-molecule observation if a drug candidate alters the normal“mechanical signature” of the target enzyme. Further, the methodsdescribed herein enable the analysis and determination of themechanism(s) by which the enzymatic dynamics are affected. The methodsutilize single molecule manipulation, detection and analysisapparatuses, to determine if and how the drug candidate modulates this“mechanical signature” in away indicative of enzymatic inhibition.

The first step in the NANO-VALID™ screening process is to choose amechanical signature that captures the functionality of the targetenzyme and is reliably measurable at the single molecule level.Exemplary signatures appropriate for specific classes of enzymes thatmay be extracted with single-molecule detection and manipulationtechnologies are discussed below in greater detail. The second step inthe NANO-VALID™ method is to experimentally determine the normal,baseline mechanical signature of the target enzyme in the absence of anyinhibitor, including a drug candidate. As discussed above, the baselinemechanical signature is determined by making a mechanical measurementusing a single molecule detection apparatus. Due to the single-moleculenature of the approach, this may involve taking ensemble averages ofseveral experiments. Exemplary technologies and apparatuses that enablesingle-molecule mechanical measurement of the dynamics of severalclasses of enzymes are discussed below.

To screen each drug candidate, the chosen mechanical measurement of thetarget enzyme is made with the same experimental techniques andapparatus and under the same conditions as those used in determining thebaseline mechanical signature, except the drug candidate is present inthe single-molecule assay. Depending on the nature of the target enzyme,it may be desirable to first incubate the target, the target'ssubstrate(s), or both, with the drug candidate for some controlledperiod of time prior to conducting this measurement.

Next, extensive signal processing is conducted to compare thecandidate-specific mechanical signature with the baseline mechanicalsignature. Exemplary variants of this analysis appropriate for variousclasses of enzymes are discussed in detail below. If no significantdeviation from the signature is detected, the candidate is rejected andnot subjected to further screening. This feature drastically reduces thetime and cost associated with drug candidate screening, testing, andvalidation, because unsuccessful drug candidates can be eliminated fromtesting much earlier in the process, much before the onset of expensiveclinical trials. This leads to much more specific drug candidates beingchosen earlier on, such that only those candidates that are more likelyto be successful make it to clinical trials. This increased selectivitycriterion early on in the drug discovery process significantly reducesthe cost and time of single molecule drug discovery processes ascompared to conventional drug validation and discovery approaches.

However, if a significant deviation from the baseline mechanicalsignature is detected, further analysis and processing is conducted toidentify potential mechanism(s) of interference. The particularembodiments of this secondary analysis appropriate for various classesof enzymes as discussed in detail below. If the identified mechanism(s)are not desirable for the disease pathway, the candidate is rejected andnot subjected to further screening. Otherwise, the candidate isconsidered for further screening and validation.

This NANO-VALID™ process is summarized in FIG. 1.

NANO-VALID™ for Polymerase Targets

A preferred embodiment of the NANO-VALID™ method that is. appropriatefor screening drug candidates that target polymerase enzymes. Theembodiment is summarized in FIG. 3. In this method embodiment, thetime-dependent position of the polymerase enzyme along the nucleic acidtemplate is always chosen at step 1 to be subsequently measured in steps2 and 3a.

Polymerases process a nucleic acid template in a primarily linearfashion. For this reason, the mechanical signature common to allpolymerases is the linear progression of the polymerase along thissubstrate. When the nucleic acid template is aligned with a fixed lineand held at constant tension, then the progression of the polymerasecorrelates simply to the position of the polymerase along this fixedline as a function of time.

In the absence of an inhibitor, a polymerase normally binds at aninitiation point, proceeds with a relatively constant velocity and thenterminates polymerization. Some minor stochastic behavior typicallyoccurs during the course of normal polymerization. These features mayinclude short pauses on the order of milli-seconds or less, shortreversals of direction on the order of less than 100 bases (20-55nanometers, depending on the experimental conditions), and variations inthe polymerization velocity on the order of a few hundred base pairs-persecond.

Shown in FIG. 6 are data demonstrating the capability to observe thedynamics of the DNA polymerase motor with an optical tweezers apparatusas it moves forwards (polymerization) and backwards (exonucleaseactivity) along a DNA template. A velocity of about 0.5 microns/mincorresponds to about 25 base-pairs polymerized/second. Without wishingto be bound by theory, it is surmised that polymerase inhibitors impactDNA polymerases (DNAp) through one of several mechanisms, each with adistinct “mechanical signature” in the plot of template length versustime, and/or the velocity of polymerization versus time. The plots canbe analyzed to identify the mechanisms. Additionally, there may be otherentirely new mechanisms, hereto unforeseen that could also be discoveredvia our methods described herein.

FIGS. 2A-2E illustrate what polymerization might look like in thepresence or absence of a drug candidate. Note that for FIGS. 2A-2Eshown, the initiation point is a y=200 μm and the polymerase is presumedto be moving from right (y=200 μm) to left (y=0 μm) in about 200minutes. When a drug candidate interferes with a polymerase target, thedynamics of the polymerase along a nucleic acid template are clearlyaffected in one of several ways, which may include: modulating the rateof polymerization, inactivating the enzyme from binding or polymerizing,altering the processivity of the enzyme, altering the binding affinityof the enzyme to the template, or altering the sequence-dependentfidelity of the enzyme. Each of these scenarios produces a mechanicalsignature distinct from normal polymerization. Thus this preferredembodiment of the NANO-VALID™ method for polymerase targets uses thetime-dependent position of the enzyme as the mechanical signature insteps 2 and 3a (see FIG. 3, NANO-VALID™ Drug Screening and Validationprocess map). FIG. 2B shows an exemplary mechanical signature that wouldindicate that a drug candidate has slowed the polymerization process.Note that the average slope of the plot, which corresponds to theaverage polymerization velocity, has lessened. Some DNA polymeraseinhibitors may work, for example, by just slowing the overall speed ofDNA replication. This trace would indicate a polymerase inhibitor drugcandidate that worked by this mechanism. FIG. 2C shows an exemplarymechanical signature that indicates that a drug candidate has inducedpremature termination, wherein the drug candidate has derailed orstalled the polymerization process before its natural completion point.Some DNA polymerase inhibitor candidates may work by such a mechanism.This trace would indicate a polymerase inhibitor drug candidate thatworked by this mechanism. FIG. 2D shows an exemplary mechanicalsignature that indicates that initiation of the polymerase is inhibitedby the drug candidate. This trace would indicate a polymerase inhibitordrug candidate that worked by this mechanism. FIG. 2E shows an exemplarymechanical signature that indicates the drug candidate induces thepolymerase enzyme to operate in an exonucleolysis mode, in which itexcises bases rather than polymerizing base-pairs. This signature wouldlikely only occur in polymerases with an active exonucleolysis, or“proof-reading” site. This trace would indicate a polymerase inhibitordrug candidate that worked by this mechanism.

To avoid false screening results, the mechanical signature used in thisembodiment of the NANO-VALID™ method should be on a length- andtime-scale that far exceeds the scale of the stochastic events of normalpolymerization. Typically, for most polymerases, the mechanicalsignature should be taken over a time window that will allow the target,under the chosen experimental conditions, to traverse a nucleic acidtemplate of at least 5000 bases or base-pairs.

In a preferred embodiment, a software algorithm to implement step 3b isutilized so that it may determined with high accuracy in step 3c if adrug candidate should be eliminated.

The stochastic nature of normal polymerization requires extraction ofthe salient features of the polymerase dynamics in steps 2 and 3a,rather than using or comparing raw data traces. The nature of the targetenzyme, as well as the desired interference mechanisms will dictatewhich salient features are extracted and analyzed. These features mayinclude: total time the motor is paused, terminal polymerizationvelocity, efficiency of termination, average velocity. Typically, forall polymerases, a low-pass filter with a cutoff frequency of 100-1000Hz will also be applied to the raw signal to filter out the effects ofthe stochastic fluctuations in the dynamics of the enzyme.

NANO-VALID™ for DNA Polymerase Targets using Template LengthMeasurements A variant embodiment of the previously describedNANO-VALID™ method that is appropriate for DNA polymerase targets thatact on a single-stranded DNA template. Each base in the single-strandedDNA template has a length of approximately 0.7 nm under standardenvironmental conditions and a constant template tension ofapproximately 0-1 pN. Each unpaired base on the template is converted bythe DNA polymerase into a base-pair with a length of approximately 0.34nm under the same environmental and constant-tension conditions. Hence,under isotension conditions, as the DNA polymerase replicates the lineartemplate, converting single-stranded DNA into double stranded DNA, thedifference in elasticity of the two states causes a shortening of theDNA strand by approximately 0.36 nm per base-pair that is polymerized ata given force. Thus, tracking the length of the DNA strand duringpolymerization under constant DNA template tension is analogous totracking the position of a polymerase along a fixed template. Therefore,in steps 2 and 3a of this method variant, the mechanical signaturedirectly obtained by the single-molecule measurement apparatus is thelength of the DNA template over time. The signal processing methodsdescribed previously for analyzing a polymerase's position and velocityin steps 3b and 3d can again be utilized in this variant method.

Apparatus for Performing NANO-VALID™ Screening of Drug Candidates

An apparatus aspect of this invention for performing the NANO-VALID™method comprises a single-molecule measurement system interfaced to apersonal computer via a rapid port (e.g., a USB port) that allows nearreal-time data-acquisition and control of the single-molecule apparatussystem. See FIGS. 9A and 9B. Via software drivers, this apparatus iscontrolled and interrogated by a custom software program that has agraphical user interface (GUI). In this embodiment, the single-moleculemeasurement system would have a receptacle for loading and addressingseveral individual samples on a single plate, for example a 96-wellmicroplate. The measurement system would integrate temperature controlsto ensure reliable and reproducible environmental conditions. Themeasurement system could comprise one or more of the following: atomicforce microscope, scanning electronic microscope, etc, etc.

To utilize the apparatus, the user would load the plate into thesingle-molecule measurement. Via the GUI, they would initialize thesingle-molecule measurement system, including any necessarycalibrations. The user would then select the mechanical signature, andany control parameters regarding the signature (for example the lengthof time that the signature is measured). Next, via the GUI, they woulddirect the single-molecule measurement system to interrogate the controlsamples to obtain the target enzyme's baseline mechanical signature. Theprecise execution of this measurement might require some manual controland input from the human user via keyboard, joystick or other computerinput device. The custom software program would then acquire thissignature, and via signal-processing routines, extract and store salientfeatures of the signal. The GUI would display the salient features,including perhaps the raw mechanical signature traces.

Next, the GUI would allow the user to traverse the remainder of theplate, to acquire a mechanical signal from each candidate; this mayentail several individual measurements. As before, this signatureacquisition step might require direct user input. At each step, the rawmechanical signature would be acquired, salient features of themechanical signature would be extracted via signal processing, stored inthe computer memory and displayed to the user, and compared to thereference baseline signal.

Apparatuses for Performing NANO-VALID™ Screening of Drug CandidatesTargeting a Polymerase

To perform the NANO-VALID™ method for polymerase targets, it isnecessary that the single-molecule measurement apparatus accuratelycapture the real-time dynamics of the polymerase along a nucleic acidtemplate. Several technologies could be utilized to accomplish thisfunctionality. For example, high-resolution fluorescent imaging coupledwith quantum-dot labeling of the polymerase could be utilized.Alternatively, atomic force microscopy could be utilized to measurethese polymerase dynamics. Magnetic or optical “tweezers” or “traps”could also be utilized for pico-Newton control of the template tensionand measurements of nanoscale displacements of the polymerase along theDNA template. Other variants could be readily envisioned.

Optical Tweezers-Based Apparatuses for Performing NANO-VALID™ Screeningof Drug Candidates Targeting a Polymerase

In a preferred embodiment, the NANO-VALID™ apparatus for polymerasetargets would utilize an optical tweezers-based single-moleculemeasurement subsystem. An optical tweezers traps particles with forcesgenerated by optical intensity gradients. Optically generated forcesstrong enough to form a three-dimensional trap can be obtained bybringing a laser beam with an appropriately shaped wavefront to a tightfocus with a high numerical aperture lens. Optical tweezers techniqueshave been used extensively for single-molecule studies of the polymerproperties of DNA and the force-dependent kinetics of biomolecularmotors, including polymerase enzymes. As a result of these extensivebiophysical studies, the experimental protocols for using opticaltweezers to track polymerase enzymes along an isotension nucleic acidtemplate are quite mature and well-known in the art (Block, et al.,Nature 348, 348-352 (1990) and Wuite et al., Nature 404: 103-106(2000)).

To track the polymerase along the template, one end of the nucleic acidtemplate is effectively immobilized, while the polymerase molecule isattached to a bead trapped in an electronically-steerable opticaltweezers apparatus. As the polymerization proceeds, the optical trap isdesigned to automatically move in order to maintain constant force onthe polymerase's bead. The dynamic position of the trap under theseisotension conditions then correlates then to the dynamics of thepolymerase along the template during polymerization. A preferredembodiment of this “Force-Feedback Optical Trapping Subsystem” isdescribed in extensive detail below. Several other variants could bereadily imagined.

FIG. 4 shows experimental variants of this embodiment; they vary intheir method of nucleic acid immobilization. In FIG. 4C the nucleic acidtemplate is attached to a dielectric bead that is held by a second,immobilized optical trap, shown at left, that exerts a strong trappingforce that is much greater than the force exerted by the polymerase onthe nucleic acid template. FIG. 4D demonstrates how the nucleic acid canbe immobilized to a rigid surface, such as a cover slip or microwellplate, via complementary functionalization of the nucleic acid templateand the rigid surface.

Methods for attaching nucleic acids and polymerases to beads or othersurfaces are well known in the art; some examples given below illustrateexemplary methods for accomplishing this task. One such methodbiotinylates the nucleic acid or polymerase via standard methods so thatit may be attached to streptavidin-coated microbeads (e.g., Bang'sLaboratories) or streptavidin-coated coverslips (e.g., XenoporeCorporation).

Optical Tweezers-Based Apparatuses for Performing NANO-VALID™ Screeningvia Single-Molecule Measurement of Nucleic Acid Template Length Thepreviously described preferred apparatus embodiment can be used toperform the variant NANO-VALID™method used for DNA polymerases along asingle-stranded DNA template. As previously discussed, this methodvariant measures the length of the DNA template, instead of directlytracking the polymerase motion. To conduct this method variant, one endof the DNA must be immobilized as before; however, the other end of theDNA template is now attached to a dielectric bead that is interrogatedby the steerable, constant-force optical trap. The enzyme remains freein solution.

In this variant method, instead of the trap moving in response to theforce of the polymerase enzyme moving along the template, it will moveto maintain constant force on the DNA template, even as the templatemolecule shortens as a result of polymerization. As discussedpreviously, this is a nearly analogous measurement. While the methodvariant requires slight differences in the sample preparation, thismethod variant requires no difference in the apparatus nor controllingsoftware. FIGS. 4A and 4B summarize the experimental variants forperforming this method variant.

Force-Feedback Optical Trapping Subsystem

FIG. 5A illustrates an exemplary design of an optical trapping systemthat maintains a constant force on a trapped bead, even as the beadexperiences other forces due to the enzymatic activity of thepolymerase. An IR laser source (A) is focused via a series of lenses andmirrors (B) to a position (xo, yo) in the sample plane. This focusposition, (xo, yo), is established by the deflection angle of a two-axisacousto-optic deflector (AOD) (L). The Gaussian beam profile at thesample slide (C) traps the bead that is attached to either a polymerase(not shown) or nucleic acid template (shown).

A quadrant photodiode or QPD, (D) detects the interference pattern ofthe IR signal scattering off the bead, and outputs the x and yperturbations of this signal. These signals are amplified (E), obtainedby a data acquisition card (DAQ), interfaced to a personal computer (G).This data is subsequently processed in a data acquisition and analysisprogram (programmed, for example, in Labview® from NationalInstruments), to determine the position of the bead relative to thecenter of the optical trap, with near-nanometer resolution and tomeasure the trapping force on the bead in picoNewtons. (See also FIG.5B)

Direct images of the bead are captured by a sensitive CCD camera (e.g.,Cooke's PixelFly) that is coupled to the light microscope (F). Customdata analysis computer programs provide nm scale position detection ofthe bead; an image analysis program provides an image of the sampleplane, including the trapped bead.

With the force feedback system operating (L, K), the force exerted bythe laser on the bead remains constant even in the presence of theenzymatic forces that act on the bead, shifting its position within thetrap. This is accomplished via a software program that determines theinstantaneous position of the bead with respect to the center of thelaser trap and compares this to a reference position (corresponding to agiven force on the trapped bead). The difference between this currentposition and the desired bead position is converted into a two channelfrequency signal (Δfx, .Δfy) that the radio frequency (RF) driver (K)inputs into the two-axis AOD crystal. This radio frequency creates astanding wave in the crystal which then diffracts the incident laserbeam. The position of the first order diffracted beam changes with theradio frequency.

Thus the laser beam can be precisely and rapidly steered by controllingthe RF input into the AOD crystal. The data acquisition programcalculates the new feedback frequency that should be generated by the RFdriver (K) to deflect the beam enough to compensate forenzymatically-driven shift in the bead's position. A record of thisoutput frequency can be post-processed to output the time evolution ofthe trap's position over the polymerization time.

The unique features of NANO-VALID™ drug validation process as comparedto conventional approaches are that the NANO-VALID™ system enables highresolution real time tracking of how the drug candidate modifies orinterferes the polymerase movement along the nucleic acid template. Thissystem is furthermore, highly integrated and automated making it easilyscalable for high throughput drug screening applications.

The methods and apparatuses described herein also provide numerouscost-related advantages over conventional drug screening and validationtechniques. As illustrated in FIGS. 8-10, the present methods andapparatus enable rapid screening, providing for an approximately115-fold reduction in labor time. In addition, the single-moleculeapproach of the present invention allows for an approximately 130-foldreduction in reagent volumes, and the reduced instrumentation cycle timeleads to an approximately 10- to 50-fold reduction in machine time.Thus, overall, the present invention provides an approximately 20- to100-fold improvement in overall cost.

EXAMPLES Method and Apparatus for Detecting Inhibition of DNAReplication Using Dynamics of Template Length

In this example, we illustrate a method and apparatus for detecting theinhibition of DNA replication via single-molecule measurement of the DNAtemplate length. In this example, we seek to screen for drugs thatinhibit DNA polymerization through one of two mechanisms: interferenceof polymerase binding, or ultra-low processivity. FIG. 2A shows anexpected normal signature, while FIG. 2B shows a result expected from aneffective drug candidate. The apparatus used for single-moleculemeasurements comprises a force-feedback optical trapping subsystem, aspreviously described. The experimental setup for this mechanicalsignature measurement is shown in FIGS. 5A and 5B.

The interior surface of an optically transparent multi-well microplateis coated with streptavidin according to standard methods. A sample ofbiotinylated ssDNA template with a length exceeding 5 kb is prepared andsuspended in a buffered solution. A DNA primer is also designed that caninitiate polymerization. A suspension of streptavidin coated 0.5-1micron beads (available from Bang's Laboratories) is prepared anddistributed among the wells; subsequently a solution of the ssDNAtemplate is also distributed among the wells. The plate is incubated forapproximately 30 minutes for the streptavidin-biotin bonding to occursuch that a sufficient population of DNA tethers are formed, which haveone end of the nucleic acid attached to the microplate, the other to astreptavidin-coated bead. The drug candidates are distributed among thewells as follows: each well contains at most one candidate; eachcandidate is added to N wells to provide redundancy. A set of C wells donot have any candidate added so that they may be used as controlsamples.

The optical tweezers apparatus is initiated and calibrated from thegraphical user interface. The microwell plate is loaded into the opticaltweezers apparatus, such that its bottom is flush with the focal planeof the optical trap. The GUI is used to set the interrogation time andthe trapping force to a desired value from 0-35 pN. The GUI also is usedto identify which candidate (if any) is present in each microwell.

Input is provided to the GUI to drive the microwell plate so that theoptical tweezers beam is interrogating the first control microwell.Using a joystick and the CCD image of the sample plane, a suitable beadis trapped manually. The force-feedback system is engaged andimmediately thereafter, a buffered suspension of DNA primer, dNTPmixture and the target DNA polymerase is added to this single microwell.This could likewise be applied to RNA polymerase and Reversetranscriptase. The force-feedback system is allowed to run to track theposition of the trap during the chosen time interval; these results arestored in memory so that they are addressable and identifiable both bythe well address and as a control result. This process is repeated forthe remainder of the control wells. From the GUI, a program is run toanalyze the multiple control samples in order to extract several salientfeatures common to the polymerase target. First, a selection ofdifferent low-pass filters, with cutoff frequencies in the range of100-1000 Hz, are applied to these controls, and the results displayed sothat the user may select the filter with a cutoff frequency roe thatgives the highest level of signal smoothing while maintaining theintegrity of the overall dynamics of the target polymerase.

Once filtered, a numerical differentiation scheme determines theinstantaneous velocity at each point. Next, nearest-neighbor averagingof the velocity is determined for different window-sizes, ranging fromthe entire acquisition window (i.e., t=[0:T]), down to a windowcorresponding roughly to 100 base-pairs (windows of approximately 1second length, depending on the velocity of the polymerase).

Input is provided to the GUI to drive the microwell plate so that theoptical tweezers beam is interrogating the first non-control microwell.Using a joystick and the CCD image of the sample plane, a suitable beadis trapped manually. The force-feedback system is engaged andimmediately thereafter, a buffered suspension of DNA primer, dNTPmixture and the target DNA polymerase is added to this single microwell.The force-feedback system continues to track the position of the trapduring the chosen time interval; these results are stored in memory sothat they are addressable and identifiable both by the well address, aswell as the drug candidate present. This process is repeated for theremainder of the control wells.

All of the obtained results are filtered with a low-pass filter withcutoff frequency roe. With the GUI, the results are numericallydifferentiated, and nearest-neighbor velocity plots are calculated forthe same time-scales as for the controls. Each drug candidate isconsidered in turn. Substantially non-zero velocity over any time scale,for any one of the N samples would indicate that the drug candidate hadnot interfered reliably or efficiently with the DNA replication process.For that reason, those candidates showing a substantially non-zerovelocity (e.g., 1% of the average velocity of all of the controls) overany time scale, for any of the N samples, are rejected.

Method and Device for Detecting Early Termination of RNA PolymeraseTargets Using Dynamics of Polymerase Along DNA Strand

In this example, we illustrate a method and apparatus for detecting theearly termination of RNA transcription by an RNA polymerase. In thisexample we are screening for drug candidates that induce earlytermination of RNA polymerization after initiation at a specific site.FIG. 2A shows an expected normal signature, while FIG. 2B shows a resultexpected from an effective drug candidate. The apparatus used forsingle-molecule measurements is a dual beam optical tweezers apparatusthat comprises both a force-feedback optical trapping subsystem and ahigher-power steerable optical trap.

A sample of double stranded (ds) DNA is prepared via standard methods tocomprise both a stalled transcription complex comprising a biotin tag,as well as biotin tag on the downstream end of the DNA (Neuman, K. C.,et. Al, Cell, 115: 437-447, 2003). The transcription factor tag issubsequently attached to streptavidin-coated 1 micron diameterdielectric bead (e.g., Bang's Laboratories), and the DNA tag is attachedto a streptavidin-coated 0.5 micron diameter dielectric bead (e.g.,Bang's Laboratories). This forms a sample of DNA “dumbbells,” that havea bead “handle” on each end: one attached to the end of the DNA, theother to the RNA polymerase. The drug candidates are distributed amongthe wells of an optically transparent microwell plate as follows: eachwell contains at most one candidate; each candidate is added to N wellsto provide redundancy. A set of C wells do not have any candidate addedso that they may be used as control samples.

The optical tweezers apparatus is initiated and calibrated from thegraphical user interface. The microwell plate is loaded into the opticaltweezers apparatus, such that its bottom is flush with the focal planeof the optical trap. The GUI is used to set the interrogation time, T,and the trapping force of the force-feedback beam to a desired valuefrom 0-35 pN. The GUI also is used to identify which candidate (if any)is present iri each microwell, so that the microwells may besubsequently addressed by both their position and contents.

Input is provided to the GUI to drive the microwell plate so that theoptical tweezers beam is interrogating the first control microwell.Using a joystick and the CCD image of the sample plane, a dumbbell istrapped manually by trapping the larger bead in the force-feedbackoptical trap, and the smaller bead in the strong secondary trap. Theforce-feedback system is engaged and immediately thereafter, a bufferedsuspension of RNA dNTP mixture is added to this single microwell. Theforce-feedback system continues to track the position of the trap duringthe chosen time interval; these results are stored in memory so thatthey are addressable and identifiable both by the well address and as acontrol result. This process is repeated for the remainder of thecontrol wells. From the GUI, a program is run that analyzes the multiplecontrol samples in order to extract several salient features common tothe polymerase target. First, a selection of different low-pass filters,with cutoff frequencies in the range of 100-1000 Hz, are applied tothese controls, and the results displayed so that the user may selectthe filter with a cutoff frequency roe that gives the highest level ofsignal smoothing while maintaining the integrity of the overall dynamicsof the target polymerase.

Once filtered, a numerical differentiation scheme determines theinstantaneous velocity at each point. Next, nearest-neighbor averagingof the velocity is determined for different window-sizes, ranging fromthe entire acquisition window (i.e., t=[0:T]), down to a windowcorresponding roughly to 100 base-pairs (windows of approximately 1second length, depending on the average velocity of the polymerase). Theuser then selects a timepoint, To<T, via the GUI, that corresponds tothe maximum time at which polymerization should still occur (i.e., thetime beyond which polymerization should be terminated by an effectivedrug candidate).

Input is provided to the GUI to drive the microwell plate so that theoptical tweezers beam is interrogating the first non-control microwell.Using a joystick and the CCD image of the sample plane, a suitable beadis trapped manually. The force-feedback system is engaged andimmediately thereafter, a buffered suspension of RNA dNTP mixture isadded to this single microwell. The force-feedback system is allowed torun to track the position of the trap during the chosen time interval;these results are stored in memory so that they are addressable andidentifiable both by the well address, as well as the drug candidatepresent. This process is repeated for the remainder of the controlwells.

All of the obtained results are filtered with a low-pass filter withcutoff frequency roe. With the GUI, the results are numericallydifferentiated, and nearest-neighbor velocity plots are calculated forthe same time-scales as the controls. Each drug candidate is consideredin turn. If a drug candidate is reliably interfering with transcriptionin the manner desired, all of the related N samples should showsubstantially non-zero velocity after the time To. For that reason,those candidates showing a substantially non-zero velocity for any ofthe N samples (e.g., 1% of the average velocity of all of the controls)for any time window after To are rejected.

While the invention has been described in detail with reference toparticular embodiments thereof, it will be apparent to one skilled inthe art that various changes can be made, and equivalents employed,without departing from the scope of the invention.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates, which may need to be independently confirmed.

All publications cited herein are hereby incorporated by reference intheir entirety.

What is claimed is: 1.-13. (canceled)
 14. A system for measuring theinteraction of a polymerase and a polymerase substrate, comprising: (a)a sample chamber containing a bound polymerase or a bound polymerasesubstrate; (b) a radiation source configured to maintain said boundpolymerase or bound polymerase substrate substantially at a focusposition in the sample plane of said sample chamber using radiationsignal focused on said sample plane; (c) a detector configured to detectinterference patterns in radiation reflected from said bound polymeraseor said bound polymerase substrate arising from spatial deviations ofsaid bound polymerase or said bound polymerase substrate from said focusposition; (d) a computer processor configured to determine spatialdeviations of said bound polymerase or said bound polymerase substratefrom said focus position using said detected spatial deviations; and (e)a force feedback system configured to apply said determined spatialdeviations to correct said radiation signal to maintain said boundpolymerase or bound polymerase substrate substantially at said focusposition.
 15. The system of claim 14, wherein said polymerase orpolymerase substrate is bound to a bead.
 16. The system of claim 15,wherein said radiation source is a laser.
 17. The system of claim 16,further comprising a two-axis acousto-optical detector, said two-axisacousto-optical detector being configured to deflect said radiation tomaintain said bound polymerase or said bound polymerase substrate atsaid focus position.
 18. The system of claim 17, wherein said detectoris a quadrupole detector configured to detect interference patterns ofthe signal from said laser scattering off of said bound polymerase orbound polymerase substrate.
 19. The system of claim 18, furthercomprising a camera, said camera being configured to determining theposition of said bound polymerase or bound polymerase substrate.
 20. Thesystem of claim 14, further comprising a two-axis acousto-opticaldetector, said two-axis acousto-optical detector being configured todeflect said radiation to maintain said bound polymerase or said boundpolymerase substrate at said focus position.
 21. The system of claim 20,wherein said detector is a quadrupole detector configured to detectinterference patterns of the IR signal from said IR laser scattering offof said bound polymerase or bound polymerase substrate.
 22. The systemof claim 21, further comprising a camera, said camera being configuredto determining the position of said bound polymerase or bound polymerasesubstrate.
 23. A method for measuring the interaction of a polymeraseand a polymerase substrate, comprising: (a) providing a bound polymeraseor a bound polymerase substrate in a sample chamber; (b) irradiatingsaid bound polymerase or bound polymerase substrate using a radiationsource and maintaining said bound polymerase or bound polymerasesubstrate substantially at a focus position in the sample plane of saidsample chamber using a radiation signal focused on said sample planefrom said radiation source; (c) detecting interference patterns inradiation reflected from said bound polymerase or said bound polymerasesubstrate arising from spatial deviations of said bound polymerase orsaid bound polymerase substrate from said focus position; (d)determining spatial deviations of said bound polymerase or said boundpolymerase substrate from said focus position using said detectedspatial deviations; and (e) applying said determined spatial deviationsto correct said radiation signal to maintain said bound polymerase orbound polymerase substrate substantially at said focus position.
 24. Themethod of claim 23, wherein said radiation is laser radiation.
 25. Themethod of claim 24, wherein said detecting is performed using a two-axisacousto-optical detector configured to deflect said radiation tomaintain said bound polymerase or said bound polymerase substrate atsaid focus position.
 26. The method of claim 25, wherein said detectoris a quadrupole detector configured to detect interference patterns ofthe signal from said laser scattering off of said bound polymerase orbound polymerase substrate.
 27. The method of claim 26, furthercomprising determining the position of said bound polymerase or boundpolymerase substrate using a camera.
 28. The method of claim 27, furthercomprising providing a drug candidate in said sample chamber.
 29. Themethod of claim 23, wherein said detecting is performed using a two-axisacousto-optical detector configured to deflect said radiation tomaintain said bound polymerase or said bound polymerase substrate atsaid focus position.
 30. The method of claim 29, wherein said detectoris a quadrupole detector configured to detect interference patterns ofthe signal from said laser scattering off of said bound polymerase orbound polymerase substrate.
 31. The method of claim 30, furthercomprising determining the position of said bound polymerase or boundpolymerase substrate using a camera.
 32. The method of claim 31, furthercomprising providing a drug candidate in said sample chamber.
 33. Themethod of claim 32, further comprising comparing the force required tomaintain the position of said bound polymerase or bound polymerasesubstrate without said drug candidate with the force required tomaintain the position of said bound polymerase or bound polymerasesubstrate combined with said drug candidate.